| Resumo: | The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman micro-spectroscopy coupled with reverse and D2O co-labelling to study metabolic interactions in a two-species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labelling has been applied to detect initially 13C-labelled bands of ADP1 SCRS reverting back to 12C positions in the presence of 12C citrate. Raman-SIP with D2O labelling has been employed to probe metabolic activity of single cells without the need of cell replication. Our results show that E. colialone in minimal medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry-based metabolite footprint analysis suggests that putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labelling would be a useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are notreadily available. It is also found that Raman-SIPwith D2O labelling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity and interactions in microbial communities at the single cell level.
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